the afm probe - fundamentals, selection, and applications · pdf fileintroduction •...
TRANSCRIPT
The AFM Probe - Fundamentals, Selection, and Applications
Introduction
• Appropriate selection of AFM probe can be one of the most important decisions to be made towards a successful AFM experiment
• As there are many probes to chose from, this selection may appear daunting
• The purpose of this webinar is to enable the AFM researcher to select an AFM probe like an expert.
Outline
• Probes overview
• Key metrics for probe choice
• Applications based probe decisions
• Very High Resolution Imaging
• Quantitative Nanomechanical
• Fast Scanning in Air and in Fluid
• Biological Applications and Force Spectroscopy
• Nano Electrical – KPFM
• Nano Optical - TERS
• Probe Preparation and Cleaning
• Bruker AFM Website
• Conclusion
Bruker Confidential 4
Basic Operation of the SPM: Simplified Schematic
AFM Probe
Bruker Confidential 5
1: Apex on size
scale of atoms
2: Sensitivity on the
scale of atoms 3: Forces commensurate
with atomic bonds.
1: The Probe Apex: The critical factor for determining AFM resolution
• The microscope maker’s rule: you can’t visualize anything smaller than what you are interrogating it with.
• This is true for photons, electrons, and AFM and why the Apex size and geometry are so important.
10/28/2013 6
RTESPA OTESPA NCHV-A TESP-HAR TESP-SS
CDR50C MCNT-500 VITA-DM-GLA-1 DPT10 MLCT (Dec’13)
2: AFM Sensitivity: From the cantilever’s perspesctive
• Simple explanation of optical lever: smaller cantilevers deflect the spot more, creating more signal:
• However, its not so simple. Optics and detection electronics come into play:
• But the same scaling holds true, shorter levers create greater signal.
• But there is a tradeoff. . .
10/28/2013 7 Bruker Confidential
See Fukuma and Jarvis, RSI, 77 (2006)
3: Spring Constant Forces must be commensurate with surface
• As you continue to reduce length, the spring constant (k) of the lever increases to the 3rd power.
• To maintain low tip sample forces the thickness of the cantilever has to be scaled at the same rate.
• This is fundamentally why AFM cantilevers are small.
• L must be short enough to get good signal – 200um or less
• T must then be thin enough to apply a force “soft” compared to surface & tip
• See brukerprobes.com for to see probes ranging from:
• L - less than 20um to greater than 300um
• t – less than 0.2um to greater than 7um
10/28/2013 8 Bruker Confidential
Sarid, “Scanning Force Microscopy”,
ISBN 9780195344691, Oxford University Press
Cantilever Dynamics and Beyond
• For many modes resonant frequency also plays a critical role in the AFM’s speed and resolution.
• The probe’s frequency scales with geometry and in general a higher frequency is better.
• So additionally, one cannot simply scale length and thickness together for many applications.
10/28/2013 9 Bruker Confidential
SCANASYST-AIR-HR
• As cantilevers get smaller, tip mass and cantilever drag are become very important and add significant complexity to cantilever design.
Point of Order. . .
“AFM’ers” will routinely use relative terms expecting them to be interpreted in the context of the Application, not absolutely.
• Cantilever Coatings – both simply called coatings
• Front (Tip) Side Coatings: Used for electrical/conductive measurements
• Backside Coatings: Used to increase laser reflectivity. These are generally prefered, but they can cause thermal drift (bimetallic effect) or contamination in biological.
• Spring Constant – “normal levers” span 4-orders of magnitude
• In Bio Applications: Soft < 0.05 N/m , Hard > .5 N/m
• In PeakForce Tapping: Soft < 0.1N/m, Hard > 20 N/m
• In Tapping Mode: Soft < 5 N/M, Hard > 50 N/m
• Resonant Frequency – “normal levers” span
• Fluid Tapping: low < 5kHz, high > 50kHz
• Air Tapping: low < 20kHz, high > 500kHz
10/28/2013 10 Bruker Confidential
VERY HIGH RESOLUTION IMAGING
10/28/2013 Bruker Confidential 11
AFM probes for very high resolution TappingMode imaging of Mica in Water
Localize interaction with very small amplitude (<2nm)
Localize interaction with very sharp tip
• May need to ‘cherry pick’ probe
• Preserve tip: moderate spring constant (1-10 N/m)
• Use the maximum Amplitude Setpoint possible (reducing peak force)
Recommended probes
• ScanAsyst-Fluid+ or FastScan B
A.Enkelmann, Adv.Pol. Sci., 1984, 63, 91
AFM probes for very high resolution PeakForce QNM of Polydiacetylene (PDA) in Air
Peak Force Tapping provides much higher resolution than TappingMode in air & provides property maps
Localize interaction with very sharp tip
• May need to ‘cherry pick’ probe tip
• Preserve tip: low spring constant <1N/m & setpoint <500pN
Recommended probes
• ScanAsyst-Fluid+ or FastScan C b
Height Stiffness Adhesion
0.5 nm 1.4 nm
lattice defect
AFM probes for very high resolution PeakForce QNM for sub-nm resolution calcite in liquid
DMTModulus channel provides a qualitative stiffness map
• Alternate rows of atoms have significantly increased contact stiffness, rows switch over step
• Need moderate spring constant for modulus contrast
Localize interaction with very sharp tip
• May need to ‘cherry pick’ probe tip
• Preserve tip: moderate spring constant <5N/m & setpoint <5nN
Recommended probe
• ScanAsyst-Fluid+ or FastScan B
Height 2 nm
Stiffness
Data collected in collaboration with Dr. Daniel Ebeling, U. Maryland
15 10/28/2013
AFM probes for very high resolution PeakForce Tapping to Resolve the DNA Double Helix
Bruker Nano Surfaces Division
MAJOR MINOR
PeakForce Tapping images reveal corrugation corresponding to widths of major and minor grooves
• DNA loosely bound to mica surface (~1mM NiCl2) to minimize conformational effects
Challenge for high-resolution imaging – requires very low force and high sensitivity
• Short cantilever provides sensitivity
• Low spring constant, setpoint protect tip and sample from damage
Recommended Probe
• FastScan-Dx: 18um length, k~0.25N/m
AFM probes for very high resolution Guidelines for probe selection
Resolution determined by localization of interaction
• Smaller tips resolve smaller features without influence from neighboring structures
• Peak Force Tapping mode localizes interaction by default while Tapping Mode requires small amplitudes to localize force
• Lateral forces can quickly damage the tip, leading to larger interaction areas
Shorter (<125um) probes are recommended
• Shorter cantilevers are more sensitive, higher frequency, and have smaller viscous background in liquid
Spring constant should be selected to avoid tip and sample damage
• Keep peak force low (typically <500pN)
• Larger spring constants are often more stable, especially in TappingMode (less concern about adhesion), so very soft (<0.5N/m) cantilevers are not recommended
Consider cantilever resonant frequency (f0)
• For Peak Force Tapping, resonant frequency f0>10*modulation frequency
• For TappingMode performance is usually better f0>10kHz
10/28/2013 16
QUANTITATIVE NANOMECHANICAL MEASUREMENTS
10/28/2013 Bruker Confidential 17
AFM Quantitative Nanomechanics (QNM) Calculate sample properties directly from force curves
Probe selection for Nanomechanical Measurement key parameters
• Tip shape (R and/or half angle)
• Spring Constant (k)
• Resonant Frequency
• Length
• Coating
Remember: Quantitative force measurement requires calibration of spring constant and deflection sensitivity. Modulus also requires calibration of tip shape. Nominal values are not accurate.
10/28/2013 18
(ii)
~
AFM probes for QNM High resolution PF-QNM of a heat-sealed bag
Study of interphase between Tie and Sealant layers requires high resolution and good modulus contrast
• Sharp tip (R<25nm) needed to provide resolution to separately measure individual lamella
• Moderate spring constant (k~2-10N/m) needed for good match to material stiffness ~100-300MPa
• Backside coating to reduce optical interference
(a)
(b)
(a)
(c)
(b)
DMTModulus
Barrier layer
Nylon Strength & gas impermeability
Tie layer
ULDPE Preserves layer adhesion
Sealant layer
Metallocene PE/LDPE blend Adheres to itself when heated
10/28/2013 19
Recommended probe:
• TAP150A also known as MPP-12120 (k~5N/m, R~8nm, Al backside coating)
AFM probes for QNM Expanding PeakForce QNM to softer samples
Real AFM tips are not a simple sphere or cone
• Choose the deformation model (Hertz/DMT (spherical) or Sneddon (conical)) that best represents your tip shape at a given deformation depth
By choosing spring constant, tip shape, and model carefully, the widest range of properties can be studied
10/28/2013 20
Real AFM tip
Tip radius ~20nm
Tip half angle
~18° Cells
& gels
AFM probes for QNM Guidelines for probe selection
Tip shape (end radius, half angle) determines resolution
• Larger tips: shape matches models better and are more stable
• Smaller tips: resolve smaller features without influence from neighboring structures
Spring constant and tip shape: select to match sample stiffness
• Most important for modulus & deformation measurements
Resonant frequency >10*modulation freq. to avoid cantilever resonance
• Not generally an issue in air, but can be a problem in liquid with soft cantilevers
Shorter is usually better
• More sensitive, higher freq, and have smaller viscous background
• Longer cantilevers are easier to align and less prone to optical interference
10/28/2013 21
AFM probes for QNM PeakForce QNM and Force Volume of Agarose gels
10/28/2013 22
A stiffer probe would
be better here
Sneddon model works well over the biologically relevant kPa-MPa range when coupled with soft cantilever in liquid
• Relatively blunt tip provides better repeatability and is more gentle for very soft samples
• Soft cantilever (k~2-10N/m) needed for good match to material stiffness ~10-500kPa
• Backside coating to improve reflectivity of silicon nitride cantilever
• Similar results obtained with PF-QNM and FV at ramp rates from 1Hz to 500Hz
Recommended probe:
• MLCT-E (k~0.1N/m, R~20nm, half angle ~18deg, Ti/Au backside coating)
AFM probes for QNM PeakForce QNM of live cells on glass bottom Petri dish
10/28/2013 23 Bruker Nano Surfaces Division
Spring constant should match modulus
• Ecell=kPa << Eglass=GPa
• MLCT-D has k ~0.03N/m
• Good for Ecell (see cell structures)
• Too small for Eglass (out of range)
Tip Shape should match Fit Model
• MLCT has pyramidal tip
• Indentation on soft cell ≥100nm
• Indentation too deep for DMT (sphere)
• Sneddon better modulus estimation
Recommended probe for cell:
• MLCT-D (k~0.03N/m, R~20nm, half angle~35deg, Ti/Au backside coating)
Modulus image of B16 mouse carcinoma cell. Force curves show fit for DMT and Sneddon modulus. Images obtained on the BioScope
Catalyst AFM in PeakForce QNM using MLCT-D probes in fluid.
Glass Surface
DMT Modulus = 50kPa Sneddon Modulus = 37kPa
FAST SCANNING IN AIR
10/28/2013 Bruker Confidential 24
Why Fast Scanning SPM?
• Fast Scanning allows real-time study of dynamic processes at the Nanoscale
• Allow rapid collection of thousands of images for meaningful statistics
• Collect large (~25MPixel) images in reasonable time for examination of both fine detail and superstructure in a single image
AFM probes for Fast Scanning in air Guidelines for probe selection
Consider cantilever resonant frequency (f0) and quality factor (Q)
• For TappingMode, cantilever bandwidth is approximately f0/Q
• Cantilever Q depends on the fluid environment (air, liquid) and on the length of the tip
• For Peak Force Tapping, resonant frequency f0>10*modulation frequency and modulation frequency should be high enough to have at least one tap per pixel
Shorter (<50um) probes are recommended
• Shorter cantilevers are higher frequency, have smaller viscous background in liquid, and are more sensitive
Resolution determined by localization of interaction
• Smaller tips resolve smaller features without influence from neighboring structures
• Peak Force Tapping mode localizes interaction by default while Tapping Mode requires small amplitudes to localize force
• Lateral forces can quickly damage the tip, leading to larger interaction areas
Spring constant & setpoint should be selected to avoid tip and sample damage
• Keep peak force as low as possible, but larger forces may be required for good tracking
• Larger spring constants are often more stable, especially in TappingMode (less concern about adhesion), so very soft (<0.5N/m) cantilevers are not recommended
Bottom line
• PeakForce Tapping is not very fast in air. For Tapping Mode, the best probe is the FastScan A!
10/28/2013 26
10/28/2013 Bruker Nano Confidential 27
Image
Specifications:
Size: 2.2um
ScanRate: 100Hz
Pixels:256 x 256
TipV: 440um/s
Frame Rate: 2.5s
Real Time Video
Duration: ~4min
Sample Courtesy:
Dr. Jamie Hobbs
University of Sheffield
HIGH SPEED AFM – FLUID IMAGING
10/28/2013 28
10/28/2013 29
AFM Probes for HS-AFM Imaging in Fluid Imaging Dynamic Biological Processes
Choosing a probe for high-speed imaging of molecular/cellular dynamics in TappingMode in fluid. (1 of 2)
• Small or Ultra-Short Cantilever – length ≤50nm.
• Provides low spring constant probes with high resonance frequency (high bandwidth).
• Soft Cantilever/Low Spring Constant (k).
• Soft samples (modulus :~kPa-MPa range.)
• Samples weakly attached to supporting substrate.
• Typically Si3N4 cantilevers. Some made of ‘new material’ (quartz-like material).
• High Resonance Frequency – f ≥80kHz.
• Facilitates high-resolution imaging at high scan speeds (secs/frame – frames/sec).
• Traditional TappingMode probes have lower resonance frequency (eg. SNL-C f~13kHz in fluid.)
• Challenging probes to design – optimizing spring constant and resonance frequency.
• IMPORTANT: Most resonance frequencies stated by manufacturer are for operation in air. This resonance frequency will decrease 1/2 to 2/3 of this value when used in fluid.
• Use (fast) thermal tune to identify the correct resonance frequency of a probe in fluid.
Bruker Nano Surfaces Division
10/28/2013 30
AFM Probes for HS-AFM Imaging in Fluid Imaging Dynamic Biological Processes
Choosing a probe for high-speed imaging of molecular/cellular dynamics in TappingMode in fluid. (2 of 2)
• Tip Sharpness.
• Small cantilevers = Force sensitivity = Imaging force = Ability to use sharper tips (w/o damage).
• FastScan-Dx has a silicon tip (R ~8nm, ≤12nm )
Bruker Nano Surfaces Division
Images of DNA origami obtained on the FastScan-Bio AFM in TappingMode using a FastScan-Dx probe in fluid (512 pixels/image) . Sample courtesty of P. Rothemond and L. Qian, CalTech.
100nm 100nm
22Hz 43Hz
100nm 100nm
11Hz
10/28/2013 31 Bruker Nano Surfaces Division
High-Speed TappingMode in Fluid.
• Short Cantilever – length ≤50nm.
• Soft Cantilever – k ~0.1N-0.3N/m.
• Stiff enough to oscillate in fluid.
• Too stiff – damage to sample.
• High Resonance Frequency – f ≥70kHz.
• Higher f = faster imaging speeds.
• Sharp tip – small radius.
• Resolve molecular structure.
Recommended AFM Probes.
• FastScan-Dx (l ~18µm; k ~0.25N/m; f
~110kHz; R ~8nm)
AFM Probes for HS-AFM Imaging in Fluid High-Speed Imaging of DNA or DNA-Protein Dynamics
0.5fps
2.0fps
1.0fps
3.0fps
High-speed imaging of Lambda Digest DNA. Parts of the DNA strand loosely bound to the surface appear to move from frame-to-frame (circle). Images were obtained on the FastScan-Bio AFM
in TappingMode using FastScan-Dx probes in fluid.
100nm
10/28/2013 32 Bruker Nano Surfaces Division
High-Speed TappingMode in Fluid.
• Short Cantilever – length ≤50nm.
• Soft Cantilever – k ~0.1N-0.3N/m.
• Too stiff = damage to sample.
• High Resonance Frequency – f ≥70kHz.
• Higher f = faster imaging speeds.
• Sharp tip – smallest radius.
• Resolve structures on cell surface.
• Small cantilever = imaging force.
Recommended AFM Probes.
• FastScan-Dx (l ~18µm; k ~0.25N/m; f
~110kHz; R ~8nm)
AFM Probes for HS-AFM Imaging in Fluid High-Resolution and High-Speed Imaging of Cell Membrane Dynamics
High-speed imaging of the effects of CM15 (AmP) on the outer membrane of live E. coli cells. Ordered structures in the
membrane are believed to be 2D-organized porin molecules. Images were obtained at 8 sec/frame on the FastScan-Bio AFM
in TappingMode using FastScan-Dx probes in fluid.
BIOLOGICAL APPLICATIONS
10/28/2013 33
10/28/2013 34
AFM Probes for Biological Samples Molecular and Live Cell Imaging
Choosing a probe for imaging biomolecules or live cells in fluid.
• Soft Cantilever/Low Spring Constant (k)
• Soft samples (modulus :~kPa-MPa range.)
• Samples weakly attached to supporting substrate.
• Typically use Si3N4 cantilevered probes (vs. Silicon).
• Gold (Au) Coated or Non-Coated Cantilever (Backside Coating).
• Al coating (air probes) can contaminate biological samples in fluid.
• Use non-coated only if absolutely necessary (see force spectroscopy section).
• Tip Sharpness.
• Sharper tips with small half angle provide high-resolution for single molecule imaging.
• Blunter tips with larger half angle provide low pressure needed for live cell imaging.
• Silicon tips tend to be sharper (smaller tip radius/half angle) than Si3N4 tips.
Bruker Nano Surfaces Division
10/28/2013 35
AFM Probes for Molecular Imaging Contact Mode Imaging of Membrane Proteins
Bruker Nano Surfaces Division
Images of aquaporin OmpF (top) and bacteriorhodopsin (bottom) obtained on the MultiMode AFM operated in
Contact Mode using a SNL-A probe in fluid. Image courtesy of D. Mueller, ETH D-BSSE, Basel.
Contact Mode Imaging in Fluid.
• Very Soft Cantilever – low k.
• sample distortion/damage.
• Gold Coated (backside) Cantilever.
• Non-coated only if necessary (drift).
• Sharp tip – small radius.
• Resolve molecular structure.
Recommended AFM Probes.
• MSNL-C (k ~0.01N/m; R ~2nm)
• SNL-D (k ~0.06N/m; R ~2nm)
10nm 10nm
20nm
10/28/2013 36
AFM Probes for Molecular Imaging PeakForce Tapping Mode Imaging of DNA
Bruker Nano Surfaces Division
PeakForce Tapping in Fluid.
• Soft Cantilever – intermediate k.
• Stiff enough to oscillate in fluid.
• Gold Coated (backside) Cantilever.
• Sharp tip – small radius.
• Resolve molecular structure.
Recommended AFM Probes.
• ScanAsyst Fluid+ (k ~0.7N/m; R ~2nm)
• SNL-A (k ~0.56N/m; R ~2nm) Image of plasmid DNA obtained on the MultiMode AFM operated in PeakForce Tapping Mode using a ScanAsyst Fluid+ probe in fluid. Image courtesy of A. Pyne and B.
Hoogenboom, UCL, London.
10/28/2013 37
AFM Probes for Live Cell Imaging Contact Mode Imaging of Live Cells
Bruker Nano Surfaces Division
45 m
Contact Mode Imaging in Fluid.
• Very Soft Cantilever – low k.
• Sample distortion/damage.
• Gold Coated (backside) Cantilever.
• Non-coated only if necessary (drift).
• Blunt tip – larger tip radius.
• Low pressure won’t damage cell.
Recommended AFM Probes.
• MLCT-C (k~ 0.01N/m; R ~20nm)
• DNP-A (k~ 0.06N/m; R ~20nm)
• OBL (k ~0.006-0.03N/m; R ~30nm)
Image of live endothelial cells obtained on the BioScope Catalyst AFM operated in Contact Mode using an MLCT-C probe in fluid.
10/28/2013 38
AFM Probes for Live Cell Imaging PeakForce Tapping Mode Imaging Live Cells
Bruker Nano Surfaces Division
PeakForce Tapping in Fluid.
• Soft Cantilever – intermediate k.
• Stiff enough to oscillate in fluid.
• Gold Coated (backside) Cantilever.
• Sharp tip – small radius.
• Resolve molecular structure.
Recommended AFM Probes.
• DNP-A (k~ 0.56N/m; R ~20nm)
• MLCT-F (k~ 0.6N/m; R ~ 20nm)
• ScanAsyst Fluid (k~ 0.7N/m; R ~20nm)
Sneddon Modulus data overlaid on 3D topography image of live E. coli cells. Dividing cell (right) has lower modulus than single cell (left). A single flagellum is also observed. Imaging
obtained on the BioScope Catalyst AFM operated in PeakForce Tapping Mode using a DNP-A probe in fluid.
5 m
FORCE SPECTROSCOPY
10/28/2013 Bruker Confidential 39
10/28/2013 40
AFM Probes for Force Spectroscopy Localized Measurements of Modulus, Molecular Unfolding, and Binding Interactions.
Bruker Nano Surfaces Division
Choosing a probe for single point force measurements of modulus (pushing) and unfolding/unbinding (pulling). (1 of 3)
• Cantilever Spring Constant (k).
• Modulus/Indentation – based on expected sample modulus (kPa–MPa = softer probes; MPa–GPa = stiffer probes).
• Unfolding/Unbinding – based on forces being measured (pN = softer probes; nN = stiffer probes).
• Measured cantilever deflection should be within 2-3V of noncontact deflection value.
• Probe too stiff = deflection too small (below noise floor – cannot measure).
• Probe too soft = deflection too large (photodetector response non-linear at large deflections).
• Important to calculate deflection sensitivity and spring constant (thermal tune). Nominal values are not accurate.
• Note: For very soft probes sometimes easier to calculate k in air and then re-measure the deflection sensitivity in fluid (k does not change).
10/28/2013 41
AFM Probes for Force Spectroscopy Localized Measurements of Modulus, Molecular Unfolding, and Binding Interactions.
Bruker Nano Surfaces Division
Choosing a probe for single point force measurements of modulus (pushing) and unfolding/unbinding (pulling). (2 of 3)
• Coated or Non-Coated Cantilever (Backside Coating).
• Al coating (air probes).
• Au coating (fluid probes) – Al can contaminate biological samples.
• Non-coated (most probes are coated)
• Backside coating can introduce drift, especially in fluid or at elevated temp (bimetallic effect).
• Use non-coated cantilever only if absolutely necessary (can introduce other issues)
• Beware of optical interference with non-coated probes (observed in baseline of force curves).
• Also ensure adequate sum signal (force sensitivity) – typically want >1.5V.
Optical interference causes regular pattern in force curve.
10/28/2013 42
AFM Probes for Force Spectroscopy Localized Measurements of Modulus, Molecular Unfolding, and Binding Interactions.
Bruker Nano Surfaces Division
Choosing a probe for single point force measurements of modulus (pushing) and unfolding/unbinding (pulling). (3 of 3)
• Tip Shape.
• Very important for modulus measurements.
• Based on modulus fit model (indentation) eg. DMT/Hertz = sphere; Sneddon = cone.
• Tip Sharpness.
• Modulus/Indentation –blunt/dull probes minimize pressure (tip or sample damage).
• Unfolding/Unbinding – larger R increases chances of molecule attached to end of tip.
• Tip Functionalization.
• Modification of tip with chemical group (eg. CH3/COOH) or molecules/ligands.
• Cleaning probe is first step in functionalization process (refer to previous slides).
• See AN#130 “Common Approaches to Tip Functionalization for AFM-Based Molecular Recognition Measurements”.
10/28/2013 43
AFM Probes for Force Spectroscopy Optically Targeted Cell Modulus Measurements
Bruker Nano Surfaces Division
Modulus Measurements (in fluid).
• Soft Cantilever – k ~0.01-0.06N/m.
• Cells have low modulus (kPa).
• Gold Coated (backside) Cantilever.
• Non-coated if drift issues.
• Conical or Spherical Tip.
• Soft cells = indentation depth.
• Sneddon = conical, Hertz = spherical.
• Blunt Tip (or Spherical) – larger radius.
• Won’t damage cell membrane.
Recommended AFM Probes.
• Spherical (k ~0.01-0.06N/m; R ~50nm-5µm)
• MLCT-C (k ~0.01N/m; R ~20nm)
Force curves performed on live endothelial cells fluorescently labeled with a membrane potential indicating
dye (bis-oxonol). Cells were found to be stiffer in hyperpolarized state. Studies conducted on the BioScope
Catalyst AFM using spherical probes (k ~0.01N/m) in fluid. Images courtesy of H. Oberleithner, Munster, Germany.
10/28/2013 44
AFM Probes for Force Spectroscopy Single Molecule (Un)Folding Studies
Bruker Nano Surfaces Division
Molecular Unfolding Studies (in fluid).
• Soft Cantilever – k ~0.01-0.06N/m.
• Forces measured are in pN range.
• Gold Coated (backside) Cantilever.
• Non-coated if drift issues.
• Blunt Tip – larger radius (R).
• >R = chances of ‘catching’ molecules.
• Tip Functionalization.
• Au-coated tips if molecule has thiol groups.
Recommended AFM Probes.
• MLCT-C (k ~0.01N/m; R ~20nm)
• MLCT-D (k ~0.03N/m; R ~20nm)
• DNP-D (k ~0.03N/m; R ~20nm)
AFM pulling/unfolding curve for a single titin 8-mer construct. The retract portion of the curve show the classic
‘sawtooth’ pattern with ~200pN force required to unfold each of the 8 repeated subunits. Force curves obtained on
the MultiMode AFM using a MLCT-C probe in fluid.
KELVIN PROBE FORCE MICROSCOPY (KPFM)
10/28/2013 Bruker Confidential 45
KPFM Modes and Probe Selection Guide
10/28/2013 46 Bruker Nano Surfaces Division
AN140-RevA1-PeakForce_KPFM-AppNote
Spatial Resolution Geometry
10/28/2013 47 Bruker Nano Surfaces Division
L W
∆L
l
The tip needs to be sharp
Tip height and cantilever width, length:
Taller, Higher aspect ratio
Smaller Cantilever
Sensitivity Cantilever Spring Constant and Q
• Higher Q (on surface) and lower k gives higher sensitivity for KPFM measurements, this is vitally important for FM-KPFM.
• Probe must also have clean resonance peak.
10/28/2013 48 Bruker Nano Surfaces Division
Thermal Tune
Q
Q/k of Some Probes Types Other factors must also be accounted for
10/29/2013 49 Bruker Nano Surfaces Division
SCM-PIT f k Q Q/k Q(Engaged) Q/k(Engaged)
#2 65 2.344 182 78 116 49
#3 66 2.712 197 73 114 42
#4 68 2.995 195 65 120 40
AC40 f k Q Q/k Q(Engaged) Q/k(Engaged)
#2 119 0.111 42 378 20 176
#3 117 0.114 46 400 26 232
#4 123 0.101 46 451 26 259
PFQNE-AL f k Q Q/k Q(Engaged) Q/k(Engaged)
#6 311 0.689 58 84 27 39
#7 312 0.669 60 89 30 45
#8 305 0.648 59 92 35 54
ScanAsyst-Air-HR f k Q Q/k Q(Engaged) Q/k(Engaged)
#2 112 0.346 59 172 26 75
#3 117 0.335 57 172 19 57
#4 116 0.436 63 144 30 69
NANO OPTICAL MICROSCOPY
10/28/2013 Bruker Confidential 50
Tip Enhanced Raman Spectroscopy probes Application: nanoChemical identification
System specific: IRIS
Key considerations
• Feedback method
• STM = gets closer but needs conducting sample
• AFM (tuning fork) = any sample, less signal
• Material
• Etched Gold yields best field enhancement
• Silver probes promising, but less consistent
• Consistent Performance
• TERS contrast must be large AND reproducible
• NearIR Deflection Laser
• Improved Optical Access
100X tip
enhancement!
October 28, 2013
See App Note 136 for more details
TERS Probes: STM vs. AFM
October 28, 2013
• STM
• STM feedback
• Requires conductive substrate
• Optimal TERS performance
• 10X contrast
• AFM (tuning fork) - NEW
• 5X contrast
• Force feedback using tuning fork
• Works on any substrate
• Coming Soon
Prototype Tuning
Fork cartridge
Tuning Fork
Probe
Zoomed-in, showing the
etched gold wire attached
53
Examples of TERS spectra for ChemID
• Innova-IRIS with IRIS TERS-STM probes
• Consistent Performance!
• Detecting molecular films that are undetectable in micro-Raman
October 28, 2013 AFM-Raman Solutions
Methylene Blue
Extremely low power
Sensitivity – fragile samples
Nile Blue
Extreme enhancement (600!)
Outstanding probe performance
Thiophenol
No far field (shown: p/s)
Sensitivity - low Raman x-section
Consistency of Bruker TERS probes
October 28, 2013
• Making probes consistently is a costly art to master
• Our testing has shown we reach >10x enhancement on average for over 80% of the tips
0
5
10
15
20
25
30
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Contr
ast
PROBE PREPARATION AND CLEANING
10/28/2013 Bruker Confidential 55
Tip cleaning “tips” For specialized experiments
• Probe cleaning NOT a recipe for cost savings
• Main purpose is for functionalizing probes or eliminating contamination in liquid
• Only in rare circumstances (i.e. DNISP) are probes recyclable
• Pre-experiment probe prep options
• Ethanol/Isopropanol – simple, good for atomic resolution
• UV light + DI water – better for removing organics (gelpak)
• Plasma etching – good for functionalization, oxidized tip
• Acidic piranha – functionalization, warning:attacks metal
• Ultrasonication - used by some but difficult to hold tip
October 28, 2013
Example of functionalized tip
Please refer to App Note 44 for more information
A biased view of tip “recovery” Weighing your options
• Cost benefit analysis
• Can you change probes without disrupting your experiment?
• If at all possible change probes!
• Last ditch efforts if probe change is not practical
• Retract probe, run AC drive on high, run frequency sweep several times (occasionally works in fluid)
• Approach sample, while in contact increase feedback to cause tip to oscillate
• “Controlled crash”, using low setpoint
• Scan clean glass slide in contact mode
• Press (i.e. perform force curve) into soft sample such as BOPP
• Special cases
• Nanoident probes – very expensive, durable, recoverable by making forceful indentation on Gold or Rubber.
• STM probes –possible to make Nano-asperity w/ hard contact. Low success rate.
October 28, 2013
BRUKER AFM PROBES WEBSITE
October 29, 2013
Bruker AFM Probes Website: www.brukerafmprobes.com
October 29, 2013
Bruker AFM Probes Website: www.brukerafmprobes.com
October 29, 2013
Bruker AFM Probes Website: www.brukerafmprobes.com
October 29, 2013
Conclusion
October 29, 2013
• Appropriate selection of AFM probe can be one of the most important decisions to be made towards a successful AFM experiment
• Though there are many probes to chose from and this selection may appear daunting the applications requirements usually point to a clear path for probe selection.
• The Bruker website has distilled this information for the user to help make their probe selection as the AFM experts do
• The Bruker applications and Probes teams are always available to assist our customers in making the best probe selection for their needs.
© Copyright Bruker Corporation. All rights reserved.
www.bruker.com
© Copyright Bruker Corporation. All rights reserved. October 29, 2013